Previously, we developed a highly sensitive assay for viremia, capable of detecting a single copy of HIV-1 RNA in plasma. This sensitivity, capable of quantitating virions in plasma down to 0.3 copies RNA/ml or less, represents a 150-fold improvement over previous "ultra-sensitive" assays. In collaboration with colleagues at Abbott Laboratories, we have found that viremia in patients is independent of regimen, but strongly associated with pretherapy virus levels, implying that, even after 7 years of treatment, cells infected prior to therapy survive to make more virus. In collaboration with Dr. R. Siliciano, we have completed a clinical trial demonstrating the lack of additional suppression of HIV-1 viremia on intensification of standard antiretroviral therapy with either protease inhibitor or NNRTI intensification (Dinoso, et al., 2009). These studies are consistent with our recent findings (Maldarelli, et al., 2007;Palmer et al., 2008) indicating little or no active cycles of HIV-1 replication during standard drug therapy. We have now extended these studies with an additional intensification trial with the new integrase inhibitor, raltegravir. In collaboration with D. McMahon at University of Pittsburgh, we demonstrate no evidence of additional suppression by raltegravir intensification (McMahon, et al., submitted). These data confirm our results from initial intensification studies and suggest no specific additional benefit of blocking integration in reducing persistent viremia. We are collaborating with ACTG investigators (Rajesh Gandhi, PI) in a large multisite, randomized study of antiretroviral intensification that will extend our pilot studies. HVIB funds and personnel were employed to complete single copy assays and maintain databases and participate in analysis of data These natural history and interventional studies indicate that viremia during suppressive antiretroviral therapy is persistent and not further suppressible by drug intensification. As a consequence, new approaches are necessary to eradicate HIV-1 infection. To investigate alternative strategies, we are currently developing a new clinical protocol to investigate the effect of interferon alpha in suppressing persistent viremia in patients suppressed on standard antiretroviral therapy (collaboration with D. McMahon, University of Pittsburgh). In addition we are collaborating with E. Reid at University of California, San Diego, to measure effects of the proteasome inhibitor, bortezomib, on persistent HIV-1 viremia in infected individuals with suppressed viemia who are receiving bortezomib as therapy for co-morbid lymphoma (Bench to Bedside, 2008). These studies will shed light on potential new directions designed to eradicate HIV-1 infection in individuals suppressed by antiretroviral therapy. Recently, a description of apparent HIV-1 eradication was reported in an HIV-1 infected individual undergoing bone marrow transplantation for acute lymphocytic leukemia. We are collaborating with these authors (G. Hutter, Charit Universittsmedizin, Berlin, Germany) to determine whether detectable HIV-1 viremia in this patient We are also addressing the anatomic distribution of HIV-1 in infected patients prior to and following therapy. In collaboration with Dr. Gupta (U. Pitt) we are comparing the relative levels of HIV-1 in seminal and blood plasma in patients undergoing antiretroviral therapy. The results of these studies will provide important information directly releant to the current controversy regarding the infectiousness of HIV-infected partners undergoing antiretroviral therapy. The single copy analytic technique is also being utilized as a benchmark for the development of new approaches to measure HIV-1. With I. Hewlett (FDA) and L. Demeter (University of Washington), we are participating in development of europium based p24 detection assays (Bench to Bedside 2008). The same single copy assay is being used (in collaboration with Dr. Bruce Walker) to probe the levels of viremia in the rare HIV-1-infected patients who are able to control their viremia at very low levels in the absence of therapy, as well as in a well-described NIH cohort of patients with HLA-restricted HIV-1 replication (with Drs. Stephen Migueles, Mark Conners, and H. Clifford Lane) and also (in collaboration with Dr. David Margolis) to see whether a drug that activates HIV expression (i.e., valproic acid) can affect the level of persistent virus. As we investigate the nature of HIV-1 replication during suppressive therapy by investigating specific patient populations. In collaboration with Dr. M. Markowitz (Rockefeller University), we are investigating the HIV-1 viremia in patients suppressed on antiretroviral therapy who initiated antiretroviral therapy during acute HIV seroconversion. Early therapy has been associated with blunted immune responses, and these studies will determine whether levels of viremia are lower than that detected in patients with chronic HIV-1 infection. In collaboration with Drs. J. Casazza and B. Graham, (NIAID VRC), we are investigating the effect of experimental therapeutic DNA vaccination on viremia in chronically infected individuals suppressed on antiretroviral therapy. [Corresponds to Project 1 in the April 2007 site visit report of the Host-Virus Interaction Branch, HIV Drug Resistance Program]